Molecular imaging and targeted therapeutic research relies heavily on affinity ligands that can seek out and bind to particular cell surface markers. These targeting ligands allow specific populations of cells to be tracked and quantified in vivo. A targeting ligand's utility lies in its ability to direct a therapeutic or imaging moiety to the targeted cells.
A number of methods have been developed to link a ligand to its cargo, based on maleimide, N-hydroxysuccinimide, carbodiimide, and click chemistries. However, many of these suffer from poor reaction efficiencies and all of them label at random residues on the ligand. Random labeling is problematic because a poorly placed conjugate can greatly reduce a ligand's affinity for its target. Additionally, ligands may receive any number of conjugates, eliminating their ability to be quantitative or stoichiometric. A few techniques have been developed to address these problems, but they have shortcomings of their own. First, expressed protein ligation (EPL) is a technique developed to attach a short synthetic peptide with orthogonal biochemistry to the N or C-terminus of a bacterially expressed protein. Unfortunately, EPL's dependence on thioesters prohibits using the technique with proteins that contain disulfides, a significant limitation. Another problem with EPL is that the ligated and unligated proteins cannot usually be separated, requiring the cheaply expressed protein to be the limiting reactant during labeling instead of the expensive synthetic peptide.
Sortase has already been used recently for a number of protein engineering tasks, including protein purifications. By placing an LPXTG motif between SrtA and a protein of interest (SrtA is upstream of the protein in this case—i.e. at the N-terminus), a calcium triggered self-cleaving peptide can be created, leaving only an N-terminal glycine behind on the protein of interest. Another common use of SrtA involves expressing a protein of interest with a C-terminal LPXTG tag. The transpeptidase is then used to link a short peptide beginning with glycine and containing a fluorophore or other unnatural moiety to the protein. However, all reported cases to date require the addition of sortase, i.e. it is not cloned into the vector, so the sortase enzyme must be purified from the ligand, adding further inefficiency. This conjugation technique also requires the purified protein to be the limiting reactant.
Accordingly, there exists a need for improved techniques for protein purification and conjugation.